1. Field of the Invention
The present invention relates to novel methods for sequencing nucleic acid molecules. More specifically, methods are provided for sequencing nucleic acid molecules present in bacterial lysates without the need to purify nucleic acid templates from highly soluble cellular components.
2. Related Art
The Sanger-dideoxy DNA sequencing method is currently the most widely used sequencing technique (Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977), Smith, L. M. et al., Nucleic Acids Res. 13:2399-2412 (1985); Smith, L. M. et al., Nature 321:674-679 (1986), Voss, H. et al., Nucleic Acids Res. 17:2517-2527 (1989); Prober, J. M. et al., Science 238:336-341 (1987), Bergot, U.S. Pat. No. 5,366,860). The Sanger-dideoxy method combined with high-throughput, automated fluorescent sequencing machines and sophisticated DNA analysis by computers has allowed the sequencing of entire genomes (Fleischmann, R. D. et al., Science 269:496-512 (1995); Fraser, C. M. et al., Science 270:397-403 (1995); Himmelreich, R. et al., Nucleic Acids Rev. 24:4420-4449 (1996)).
The quality of the nucleic acid template used for automated fluorescent sequencing is integral to the success of the cycle sequencing reactions. The importance of high quality nucleic acid template has led to the development of both manual and robotic systems to generate nucleic acid template suitable for sequencing. However, large genome sequencing projects would benefit directly from reductions in the time and cost of nucleic acid template preparation.
Previous efforts using heat-soaked polymerase chain reaction (HS-PCR, Chen, Q. et al., BioTechniques 21:453-457 (1996)) or asymmetric polymerase chain reaction (Wilson, R. et al., BioTechniques 8:184-189 (1990)) have sacrificed data quality in order to eliminate or minimize the effort needed to purify nucleic acid template.
The present invention provides methods for sequencing nucleic acid molecules, referred to as Direct Bacterial Lysate Sequencing (DBLS), without the need to purify nucleic acid templates from cellular components present in bacterial cell lysates.
In one general aspect the invention provides methods for sequencing nucleic acid templates comprising the steps of lysing bacterial host cells to produce a bacterial cell lysate containing the nucleic acid template and sequencing the nucleic acid template present in the bacterial cell lysate. In a more specific aspect the nucleic acid template present in the bacterial lysate is not amplified by polymerase chain reaction (PCR) prior to sequencing. In another specific aspect the nucleic acid template is not separated from highly soluble cellular components (e.g., sheared low molecular weight chromosomal DNA, small RNA molecules, salts, nucleotides, nucleoside monophosphates, nucleoside diphosphates, nucleoside triphosphates, amino acids, small peptides, and small carbohydrates) prior to sequencing. In yet another specific aspect the method includes culturing the bacterial host cells containing the nucleic acid template prior to lysis.
The invention further provides methods for sequencing nucleic acid templates comprising the steps of lysing the bacterial host cells to produce a bacterial cell lysate containing the nucleic acid template and sequencing the nucleic acid template using a sequencing reaction and detectable label to detect the products of the sequencing reaction. In a more specific aspect the detectable label used for sequence product detection comprises a fluorescent dye.
Generally, the method used for sequencing the nucleic acid template will be one which employs a detection method for identifying a detectable label which allows for the detection of low concentrations of sequencing reaction products. Such detectable labels include high intensity fluorescent dyes, infrared dyes, and radioactive labels. High intensity fluorescent dyes include the d-rhodamine and fluorescein/d-rhodamine dyes having the structures shown in FIGS. 6A-6F, FIGS. 7A-7D, FIGS. 8A-8D, and FIGS. 9A-9D.
The nucleic acid templates present in the cell lysate may be separated from cellular debris and precipitated material by one method or a combination of methods (e.g., centrifugation) prior to sequencing. Further, these nucleic acid templates will generally be sequenced using thermal cycle sequencing, but, in an alternative aspect, may be amplified prior to sequencing. When PCR is used to amplify the nucleic acid templates, the amplified templates will generally be present in concentrations high enough so that any number of art known methods for sequencing nucleic acid may be used (e.g., thermal cycle sequencing, Maxam-Gilbert, Sanger sequencing, and exonuclease digestion sequencing).
More specifically, the invention relates to methods for directly sequencing nucleic acid templates in bacterial lysates in which cells of individual bacterial colonies, each harboring a vector containing identical nucleic acid templates, are removed from a plate containing solidified cultures and used to inoculate a liquid culture medium. The bacterial host cells in the liquid culture medium are allowed to multiply for a period of time, after which these cells are pelleted by centrifugation and washed to remove residual culture medium. The washed cells are then resuspended in an aqueous solution (e.g., water) and incubated at 90-98xc2x0 C. for 5-30 minutes to induce cell lysis. The samples are then cooled either on ice or to room temperature and centrifuged. After centrifugation, the resulting lysates are used in cycle sequencing reactions. Preferably, the bacterial host cells are E. coli and are cultured in Luria broth or Terrific broth (TB) (Difco Laboratories).
In an additional aspect the nucleic acid template concentration present during the sequencing reaction is between about 15 ng and about 2 xcexcg. As explained in detail below, a number of factors can be varied to change the amount of nucleic acid template present during the sequencing reaction (e.g., the bacterial host cell concentration from which the bacterial cell lysate is generated can be altered and the bacterial host cells can be lysed in varying volumes of lysis solutions).
In another aspect the present invention provides methods for sequencing nucleic acid templates comprising lysing bacterial host cells containing the nucleic acid templates to produce a cell lysate and forming in the cell lysate mixture of first, second, third, and fourth classes of polynucleotides. The polynucleotides in the first class have a 3xe2x80x2-terminal dideoxyadenosine, the polynucleotides in the second class have a 3xe2x80x2-terminal dideoxycytidine, the polynucleotides in the third class have a 3xe2x80x2-terminal dideoxyguanosine, and the polynucleotides in the fourth class have a 3xe2x80x2-terminal dideoxythymidine. Further, the polynucleotides in each of these classes are labeled with at least one detectable label. In one related aspect the nucleic acid template is not purified from highly soluble cellular components (e.g., sheared low molecular weight chromosomal DNA, small RNA molecules, salts, nucleotides, nucleoside monophosphates, nucleoside diphosphates, nucleoside triphosphates, amino acids, small peptides, and small carbohydrates) which are released from the bacterial host cells upon lysis. In another related aspect the nucleic acid template is not amplified by polymerase chain reaction prior to forming the mixture of polynucleotides. As above, the detectable label can be a fluorescent dye.
In yet another aspect the present invention provides methods for sequencing nucleic acid templates comprising hybridizing primers to the nucleic acid template, forming mixtures comprising the nucleic acid template, deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate, DNA polymerase, and dideoxynucleosides, incubating the mixtures under conditions suitable for the synthesis of populations of DNA molecules complementary to portions of the nucleic acid template, and separating the synthesized DNA molecules by size so that at least a part of the nucleotide sequence of the nucleic acid template can be determined. Generally, the nucleic acid template which is sequenced is present in a cell lysate that is prepared by lysing bacterial host cells containing the nucleic acid template. Further, the nucleic acid template is generally not purified from highly soluble cellular components (e.g., sheared low molecular weight chromosomal DNA, small RNA molecules, salts, nucleotides, nucleoside monophosphates, nucleoside diphosphates, nucleoside triphosphates, amino acids, small peptides, and small carbohydrates) released from the bacterial host cells upon lysis and is not amplified by PCR prior to forming the mixture of polynucleotides. Again, as above, the detectable label can be a fluorescent dye.
The present invention also provides methods for sequencing nucleic acid molecules of cDNA and genomic libraries. According to these methods, as above, bacterial host cells containing specific cloned nucleic acid sequences are isolated, a cultured and then lysed. The nucleic acid templates present in the bacterial cell lysate will generally be either sequenced using thermal cycle sequencing or amplified by PCR and then sequenced by art known methods (e.g., thermal cycle sequencing, Maxam-Gilbert, Sanger sequencing, and exonuclease digestion sequencing). Alternatively, methods for sequencing nucleic acid templates other than thermal cycle sequencing could be used when templates are not amplified by PCR, if the detection method used is one with high sensitivity (e.g., detection methods which use high intensity fluorescent dyes).
In another aspect the invention provides methods for high-throughput sequencing of nucleic acid templates. The DBLS methods of the invention are readily adaptable for high-throughput sequencing of nucleic acid templates. Such methods are useful when large numbers of nucleic acid templates are sequenced as, for example, when the sequence of the genome of an entire organism is sought.
The high-throughput sequencing methods of the invention involve the automated processing of one or more steps of the DBLS methods. For example, individual colonies comprising host cells which contain identical nucleic acid templates may be identified on petri dishes by optical methods and automatically transferred into individual wells of a multi-well culture plate for further culture. Additionally, after a colony has been cultured to a suitable optical density, a mechanical device may be used to remove the culture medium from each well of the multi-well plate, add reagents to each well of the wells, and/or shift the incubation temperature to induce cell lysis.